CN114990064B - Hematopoietic stem cells, preparation method and application thereof - Google Patents
Hematopoietic stem cells, preparation method and application thereof Download PDFInfo
- Publication number
- CN114990064B CN114990064B CN202210816611.8A CN202210816611A CN114990064B CN 114990064 B CN114990064 B CN 114990064B CN 202210816611 A CN202210816611 A CN 202210816611A CN 114990064 B CN114990064 B CN 114990064B
- Authority
- CN
- China
- Prior art keywords
- hematopoietic stem
- stem cells
- concentration
- differentiation step
- stage differentiation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/145—Thrombopoietin [TPO]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/165—Vascular endothelial growth factor [VEGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2303—Interleukin-3 (IL-3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Diabetes (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a hematopoietic stem cell, a preparation method and application thereof; hematopoietic stem cells include at least one of those whose surface molecules are labeled cd34+cd45-cd90+ and those whose surface molecules are labeled cd34-cd45-cd90+cd49f+cd133+cxcr4+; the preparation method of the hematopoietic stem cells comprises the following steps: a digestion step; a first stage differentiation step: performing suspension culture on the EB corpuscles by a culture system comprising cytokines bFGF, BMP4 and VEGF; a second stage differentiation step: continuing suspension culture of the EB corpuscles by a culture system comprising cytokines bFGF, VEGF and SCF; a third stage differentiation step; the method realizes in vitro induced differentiation of human Induced Pluripotent Stem Cells (iPSCs) to generate high-purity and high-potential hematopoietic stem cells by standard and quantitative means, and has controllability and operability.
Description
Technical Field
The invention relates to a hematopoietic stem cell, a preparation method and application thereof, belonging to the technical fields of cell biology and stem cytology.
Background
Hematopoietic Stem Cell (HSC) transplantation is an effective intervention and treatment means for various clinical diseases, but currently there is still no method for effectively amplifying primary autologous and allogenic hematopoietic stem cells in vitro, and the in vitro amplification of primary hematopoietic stem cells also causes replicative senescence of hematopoietic stem cells, which greatly limits clinical application of hematopoietic stem cells.
At present, research and development industry has begun to explore the adoption of human Induced Pluripotent Stem Cell (iPSC) to differentiate to generate hematopoietic stem cells, and the strategy can well solve the dilemma of in vitro expansion of primary hematopoietic stem cells, but the current system for in vitro induced differentiation of iPSC to generate HSC is imperfect, and has the following defects: 1) The induction system used in the early stage depends on the trophoblast cells, so that the standards are difficult to unify, and the trophoblast cells can be possibly doped in clinical application; 2) The time for inducing differentiation is too long, and the induction period of part of the system is as long as 3 weeks or more; 3) Because the cytokine combinations used for inducing differentiation are different, HSC (HSC) stem property induced by a part of the system is not high, and a certain proportion of CD45 positive cells are presented, or a plurality of cell populations with different differentiation degrees are doped; 4) Some systems use adherent ipscs to induce HSCs in one step, while iPSC clone size and density are closely related to HSC differentiation maturation rate, so these systems are difficult to normalize. The above problems are also apparent with kits for inducing differentiation of ipscs to produce HSCs, including the currently widely marketed Stemcell company.
The prior art methods allow expansion of hematopoietic stem cell precursors including mesodermal cells and hematopoietic endothelial cells at an initial stage by direct adherent differentiation of ipscs (Hemogenic endothelium). However, the efficiency percentage of the method for producing the hematopoietic stem cells from the hematopoietic endothelial cells is not high, because the precursor cells mainly proliferate in the early stage of induced differentiation under the condition of adherent growth, can obviously inhibit the directional differentiation of the precursor cells, and when the cell density is high during adherent growth, the adjacent cells can be inhibited by NOTCH signal paths, so that the differentiation of the hematopoietic stem cells is inhibited. The hematopoietic stem cells induced by the method are CD45+, similar to the hematopoietic stem cells derived from umbilical cord blood, can be rapidly differentiated into downstream immune cell groups, but probably belong to hematopoietic stem cells in the later differentiation stage, do not have the ability of keeping self-renewal in vivo for a long time, and cannot or cannot not efficiently reconstruct the comprehensive hematopoietic function of bone marrow.
Disclosure of Invention
In order to overcome the shortcomings of the prior art, a first object of the present invention is to provide a hematopoietic stem cell which is capable of self-renewal over a long period of time, and which is excellent in stem property for efficient differentiation.
The second object of the present invention is to provide a method for preparing the hematopoietic stem cells, which realizes in vitro induced differentiation of human induced pluripotent stem cells ipscs to produce high purity and high potential hematopoietic stem cells by standard, quantitative means, with controllability and operability.
It is a third object of the present invention to provide the use of hematopoietic stem cells for the preparation of a formulation and for in vivo bone marrow reconstruction.
The first object of the invention can be achieved by adopting the following technical scheme: a hematopoietic stem cell comprising at least one of a hematopoietic stem cell labeled with a surface molecule of cd34+cd45-cd90+ and a hematopoietic stem cell labeled with a surface molecule of cd34-cd45-cd90+cd49f+cd133+cxcr4+.
The second object of the invention can be achieved by adopting the following technical scheme: a method of producing hematopoietic stem cells comprising:
digestion step: digesting the cloned iPSC to obtain single cells, and inducing to generate embryoid bodies;
a first stage differentiation step: performing suspension culture on embryoid bodies by using a culture system comprising cytokines bFGF, BMP4 and VEGF;
a second stage differentiation step: continuously suspending and culturing embryoid bodies which complete the first-stage differentiation step by using a culture system comprising cytokines bFGF, VEGF and SCF;
a third stage differentiation step: continuing suspension culture of embryoid bodies which complete the second stage differentiation step by a culture system comprising cytokines SCF, IL-3, G-CSF, FLT3-L and TPO; hematopoietic stem cells with surface molecular markers of CD34+CD45-CD90+ are obtained.
Further, in the digestion step, iPSC is grown to 70-80% confluence for digestion.
Further, in the first stage differentiation step, the concentration of bFGF in a culture system is 10-200ng/mL; the concentration of BMP4 is 10-200ng/mL; VEGF concentration was 10-200ng/mL.
Further, in the second stage differentiation step, the concentration of bFGF in the culture system is 10-200ng/mL; VEGF concentration is 10-200ng/mL; the concentration of SCF is 10-200ng/mL.
Further, in the third stage differentiation step, the concentration of SCF in the culture system is 10-200ng/mL; IL-3 concentration is 10-200ng/mL; G-CSF at a concentration of 10-200ng/mL; the concentration of FLT3-L is 10-200ng/mL; TPO concentration is 10-200ng/mL.
Further, the third stage differentiation step further comprises: after suspension culture, the embryoid bodies are subjected to adherence culture in an untreated low-adherence culture dish pre-coated with laminin by a culture system comprising cytokines SCF, IL-3, G-CSF, FLT3-L and TPO; hematopoietic stem cells with surface molecular markers of CD34-CD45-CD90+CD49f +CD133+CXCR4+ are obtained.
Further, in the first stage differentiation step, the second stage differentiation step and the third stage differentiation step, the culture system further comprises a wnt signaling pathway activator.
Further, the wnt signaling pathway activator was CHIR99021.
The third object of the invention can be achieved by adopting the following technical scheme: a preparation of hematopoietic stem cells, the preparation comprising at least one of hematopoietic stem cells labeled with surface molecules cd34+cd45-cd90+ and hematopoietic stem cells labeled with surface molecules cd34-cd45-cd90+cd49f+cd133+cxcr4+.
Compared with the prior art, the invention has the beneficial effects that:
1. the hematopoietic stem cells of the invention have excellent stem property and can self-renew for a long time;
2. the preparation method of the hematopoietic stem cells realizes in-vitro induced differentiation of human Induced Pluripotent Stem Cells (iPSC) to generate high-purity and high-potential hematopoietic stem cells by standard and quantitative means, has controllability and operability, and overcomes the problems of unstable induction effect and uneven cell maturation time caused by floating clone size of each plating cell in the adherent differentiation method;
3. the preparation method of the hematopoietic stem cells can repeatedly induce differentiation, set specific conditions, successfully and efficiently obtain high-quality hematopoietic stem cells, and the obtained hematopoietic cells have different characteristics of expressing CD34 and reflect different pluripotency of the stem cells, so that the efficiency of inducing the hematopoietic stem cells is greatly improved, and the yield is improved;
4. the application of the hematopoietic stem cells of the invention can prepare a preparation for in vivo bone marrow reconstruction.
Drawings
FIG. 1 is a diagram of the production of EB bodies of uniform size by centrifugation of example 1;
FIG. 2 is the Day8 EB minibody of example 1;
FIG. 3 is a Day12 EB minibody of example 1
FIG. 4 is a surface molecular phenotype identification of mature hematopoietic stem cells of example 1;
FIG. 5 is the Day8 EB minibody of example 2;
FIG. 6 is a molecular phenotypic analysis of the cell wall-attached differentiated cells of Day10 EB, example 2;
FIGS. 7-12 are CFU colony generation experiments of example 3;
FIG. 13 shows the results of the bone marrow hematopoietic reconstitution experiment of example 4.
Detailed Description
The invention will be further described with reference to the accompanying drawings and detailed description below:
a method of producing hematopoietic stem cells comprising:
digestion step: digesting iPSC which is grown to 70-80% of confluence to obtain single cells, preparing single cell suspension, adding single cell suspension (containing 10000-50000 cells) into a round bottom micro-pore plate, centrifuging at a low rotating speed, culturing in an incubator for 24 hours, and inducing to generate Embryoid Bodies (EBs);
a first stage differentiation step: performing suspension culture on the EB corpuscles by a culture system comprising cytokines bFGF, BMP4 and VEGF; the concentration of bFGF in the culture system is 10-200ng/mL; the concentration of BMP4 is 10-200ng/mL; VEGF concentration is 10-200ng/mL; first stage differentiation induces hematopoietic endothelial precursor cell production; in the culture system, the addition of bFGF is beneficial to the stabilization of EB corpuscles, and the simultaneous addition of BMP4 and VEGF is beneficial to the rapid differentiation of cells in the EB corpuscles to hematopoietic precursor cells;
a second stage differentiation step: continuously suspending and culturing the EB corpuscles which complete the first stage differentiation step by a culture system comprising cytokines bFGF, VEGF and SCF; the concentration of bFGF in the culture system is 10-200ng/mL; VEGF concentration is 10-200ng/mL; the concentration of SCF is 10-200ng/mL; preliminary induction of hematopoietic stem cell HSC production; wherein the addition of VEGF can continuously promote the differentiation of hematopoietic endothelial cells, while the addition of SCF can induce the differentiation of hematopoietic endothelial cells into hematopoietic stem cells;
a third stage differentiation step: continuing suspension culture of the EB corpuscles which complete the second stage differentiation step by a culture system comprising cytokines SCF, IL-3, G-CSF, FLT3-L and TPO; obtaining hematopoietic stem cells with surface molecular markers of CD34+CD45-CD90+;
the EB corpuscles after suspension culture can also be placed in an untreated and low-adhesion culture dish which is pre-coated by fibronectin, and the culture system comprising cytokines SCF, IL-3, G-CSF, FLT3-L and TPO is used for adhesion culture; obtaining hematopoietic stem cells with surface molecular markers of CD34-CD45-CD90+CD49f +CD133 +CXCR4+;
the concentration of SCF in the culture system is 10-200ng/mL; IL-3 concentration is 10-200ng/mL; G-CSF at a concentration of 10-200ng/mL; the concentration of FLT3-L is 10-200ng/mL; TPO concentration is 10-200ng/mL;
the stimulation sequence, time and stimulation dose of the cell by the cell factor can lead the differentiation to reach the optimal effect; realizing the acquisition of high-purity hematopoietic stem cells with surface molecular markers of CD34+CD45-CD90+ and hematopoietic stem cells with surface molecular markers of CD34-CD45-CD90+CD49f+CD133+CXCR4+ in the same system;
NCBI gene numbering related to cytokines is as follows:
bFGF Gene ID:2247;BMP4 Gene ID:652;VEGF Gene ID:7422;SCF Gene ID:4254;IL-3 Gene ID:3562;G-CSF Gene ID:1440;FLT3-L Gene ID:2323;TPO Gene ID:7066。
hematopoietic stem cells include at least one of those whose surface molecules are labeled cd34+cd45-cd90+ and those whose surface molecules are labeled cd34-cd45-cd90+cd49f+cd133+cxcr4+; is used for preparing preparation and can be used for in vivo bone marrow reconstruction.
Example 1:
a method of producing hematopoietic stem cells comprising:
-1Day digestion step: digesting iPSC which is cloned and grown to 70-80% of confluence into single cells by Ackutase, washing and re-suspending by mTESR culture medium to prepare single cell suspension, adding single cell suspension (containing 20000 cells) into a round bottom 96-well plate, centrifuging for 1min at 100xg, generating EB corpuscles with uniform size, culturing in an incubator for 24h as shown in figure 1, and inducing to generate the EB corpuscles;
day0-Day2 first stage differentiation step: transferring the formed EB corpuscles into a 24-well plate with low adhesion, placing 5 EB corpuscles in one well, and performing suspension culture on the EB corpuscles for 48 hours in a culture system comprising 1×ITS-X, cytokines bFGF, BMP4 and VEGF; the concentration of bFGF in the culture system is 10-200ng/mL; the concentration of BMP4 is 10-200ng/mL; VEGF concentration is 10-200ng/mL;
day2-Day4 second stage differentiation step: removing the culture system of the first stage differentiation step, and continuing suspension culture of the EB corpuscles completed with the first stage differentiation step for 48 hours by using a culture system comprising a Stemline II, 1 xITS-X, cytokines bFGF, VEGF and SCF; the concentration of bFGF in the culture system is 10-200ng/mL; VEGF concentration is 10-200ng/mL; the concentration of SCF is 10-200ng/mL;
day4-Day6 third stage differentiation step: removing the culture system of the second stage differentiation step, and continuing suspension culture for 48h on the EB corpuscles which complete the second stage differentiation step by using a culture system comprising Stemline II, 1 xITS-X, cytokines SCF, IL-3, G-CSF, FLT3-L and TPO; the concentration of SCF in the culture system is 10-200ng/mL; IL-3 concentration is 10-200ng/mL; G-CSF at a concentration of 10-200ng/mL; the concentration of FLT3-L is 10-200ng/mL; TPO concentration is 10-200ng/mL;
day6-Day12: continuing to adopt a third-stage culture system, carrying out half liquid exchange every 48h, gradually dissociating and shedding hematopoietic stem cells in Day8 EB corpuscles as shown in figure 2, gradually dissociating and shedding hematopoietic stem cells in Day12 EB corpuscles, forming single cells as shown in figure 3, and obtaining the hematopoietic stem cells with surface molecular markers of CD34+CD45-CD90+ as shown in figure 4.
Example 2:
a method of producing hematopoietic stem cells comprising:
-1Day digestion step: digesting iPSC which is cloned and grown to 70-80% of confluence into single cells by Ackutase, washing and re-suspending by mTESR culture medium to prepare single cell suspension, adding single cell suspension (containing 20000 cells) into a round bottom 96-well plate, centrifuging for 1min at 100xg, culturing for 24h in an incubator, and inducing to generate EB small bodies;
day0-Day2 first stage differentiation step: transferring the formed EB corpuscles into a 24-well plate with low adhesion, placing 5 EB corpuscles in one well, and performing suspension culture on the EB corpuscles for 48 hours in a culture system comprising 1×ITS-X, cytokines bFGF, BMP4 and VEGF; the concentration of bFGF in the culture system is 10-200ng/mL; the concentration of BMP4 is 10-200ng/mL; VEGF concentration is 10-200ng/mL;
day2-Day4 second stage differentiation step: removing the culture system of the first stage differentiation step, and continuing suspension culture of the EB corpuscles completed with the first stage differentiation step for 48 hours by using a culture system comprising a Stemline II, 1 xITS-X, cytokines bFGF, VEGF and SCF; the concentration of bFGF in the culture system is 10-200ng/mL; VEGF concentration is 10-200ng/mL; the concentration of SCF is 10-200ng/mL;
day4-Day6 third stage differentiation step: removing the culture system of the second stage differentiation step, and continuing suspension culture for 48h on the EB corpuscles which complete the second stage differentiation step by using a culture system comprising Stemline II, 1 xITS-X, cytokines SCF, IL-3, G-CSF, FLT3-L and TPO; the concentration of SCF in the culture system is 10-200ng/mL; IL-3 concentration is 10-200ng/mL; G-CSF at a concentration of 10-200ng/mL; the concentration of FLT3-L is 10-200ng/mL; TPO concentration is 10-200ng/mL;
day6-Day10: the EB corpuscles after suspension culture are placed in an untreated and low-adhesion culture dish which is pre-coated by laminin, and are subjected to adherence culture by adopting a third-stage culture system, liquid exchange is carried out every 48 hours, day8 and hematopoietic stem cells migrate out of the EB corpuscles and further proliferate, expand and differentiate, as shown in figure 5, and the hematopoietic stem cells with surface molecular markers of CD34-CD45-CD90+CD49f+CD133+CXCR4+ are obtained, as shown in figure 6.
Example 3:
to verify the functionality of the differentiated hematopoietic stem cells, it was demonstrated that they successfully differentiated to generate downstream immune cell populations, and we therefore performed colony formation experiments with differentiated mature hematopoietic stem cells. The kit used in this experiment was Starter Kit for Methocult from stemcell TM H4434。
1) After the completion of the washing of the mature hematopoietic stem cells of example 1 with PBS, they were resuspended in IMDM containing 2% v/v FBS, wherein each 100uL of the resuspension contained 5X10 4 And hematopoietic stem cells.
2) 2mL of complete MethoCurt was added to a 15mL centrifuge tube TM The culture broth was added with 200uL of hematopoietic stem cell suspension and thoroughly mixed.
3) 2.2mL of the well-mixed cell mixture was equally distributed to two 35mm cell culture dishes, and a lid was closed, 1.1mL of the cell mixture per dish.
4) Two 35mm cell culture dishes were placed in a 10mm cell culture dish, an empty 35mm dish was placed again, 1mL of sterilized water was added, the dish was placed open, and finally a 10mm cell culture dish was covered and placed in a cell culture box for culturing.
5) After 14 days, the formation of various immune cell clones, the ability to differentiate to produce different blood cell populations, was observed under a microscope and is shown in FIGS. 7-12.
Example 4:
to verify the hematopoietic capacity of differentiated hematopoietic stem cells in vivo, we further performed bone marrow reconstitution by immunodeficient mice.
1) According to example 1, the induction of mature hematopoietic stem cells;
2) Immunodeficient mice NOD/ShiLtJGpt purchased for 4 weeks in sizePrkdc em26cd52 Il2rg em26cd22 Gpt and are kept in a sterile environment;
3) The immunodeficient mice were subjected to ionizing radiation at an irradiation intensity of 2.5Gray.
4) After 24h of irradiation, mice were injected 5X10 by tail vein 5 And hematopoietic stem cells.
5) After 3 months of feeding, taking mouse bone marrow cells, treating the mouse bone marrow cells by using a red blood cell lysate, and then detecting hematopoietic stem cells, hematopoietic precursor cells and mature immune cells in bone marrow by using a flow cytometry method, wherein the hematopoietic stem cells, the hematopoietic precursor cells and the mature immune cells are successfully transplanted in vivo and downstream hematopoietic differentiation is completed, and downstream precursor cells and mature immune cells are generated as shown in fig. 13, wherein P1-1: CD 45-CD34+CD38-hematopoietic stem cells; p1-2: CD45-cd34+cd38+ hematopoietic stem cells; p2-1: cd45+cd38+cd34+ hematopoietic stem cells; p2-2: cd45+cd34+cd38-myeloid/lymphoid precursor cells; p3-1: CD45+CD38+CD34-mature lymphocytes.
Various other corresponding changes and modifications will occur to those skilled in the art from the foregoing description and the accompanying drawings, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Claims (10)
1. A hematopoietic stem cell, wherein the hematopoietic stem cell comprises at least one of a hematopoietic stem cell with a surface molecular marker of cd34+cd45-cd90+ and a hematopoietic stem cell with a surface molecular marker of cd34-cd45-cd90+cd49f+cd133+cxcr4+.
2. A method for producing hematopoietic stem cells, comprising:
digestion step: digesting the cloned iPSC to obtain single cells, and inducing to generate embryoid bodies;
a first stage differentiation step: performing suspension culture on embryoid bodies by using a culture system comprising cytokines bFGF, BMP4 and VEGF;
a second stage differentiation step: continuously suspending and culturing embryoid bodies which complete the first-stage differentiation step by using a culture system comprising cytokines bFGF, VEGF and SCF;
a third stage differentiation step: continuing suspension culture of embryoid bodies which complete the second stage differentiation step by a culture system comprising cytokines SCF, IL-3, G-CSF, FLT3-L and TPO; hematopoietic stem cells with surface molecular markers of CD34+CD45-CD90+ are obtained.
3. The method of producing hematopoietic stem cells of claim 2, wherein in the digestion step, ipscs are grown to 70-80% confluency for digestion.
4. The method for producing hematopoietic stem cells according to claim 2,
in the first stage differentiation step, the concentration of bFGF in a culture system is 10-200ng/mL; the concentration of BMP4 is 10-200ng/mL; VEGF concentration was 10-200ng/mL.
5. The method for producing hematopoietic stem cells according to claim 2,
in the second stage differentiation step, the concentration of bFGF in a culture system is 10-200ng/mL; VEGF concentration is 10-200ng/mL; the concentration of SCF is 10-200ng/mL.
6. The method for producing hematopoietic stem cells according to claim 2,
in the third stage differentiation step, the concentration of SCF in a culture system is 10-200ng/mL; IL-3 concentration is 10-200ng/mL; G-CSF at a concentration of 10-200ng/mL; the concentration of FLT3-L is 10-200ng/mL; TPO concentration is 10-200ng/mL.
7. The method for producing hematopoietic stem cells according to claim 2,
the third stage differentiation step further comprises: after suspension culture, the embryoid bodies are subjected to adherence culture in an untreated low-adherence culture dish pre-coated with laminin by a culture system comprising cytokines SCF, IL-3, G-CSF, FLT3-L and TPO; hematopoietic stem cells with surface molecular markers of CD34-CD45-CD90+CD49f +CD133+CXCR4+ are obtained.
8. The method for producing hematopoietic stem cells according to claim 2,
in the first stage differentiation step, the second stage differentiation step and the third stage differentiation step, the culture system further comprises a wnt signaling pathway activator.
9. The method of producing hematopoietic stem cells of claim 8 wherein the wnt signaling pathway activator is CHIR99021.
10. A preparation of hematopoietic stem cells, comprising at least one of hematopoietic stem cells with surface molecular markers cd34+cd45-cd90+ and hematopoietic stem cells with surface molecular markers cd34-cd45-cd49 f+cd133+cxcr4+.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210816611.8A CN114990064B (en) | 2022-07-12 | 2022-07-12 | Hematopoietic stem cells, preparation method and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210816611.8A CN114990064B (en) | 2022-07-12 | 2022-07-12 | Hematopoietic stem cells, preparation method and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114990064A CN114990064A (en) | 2022-09-02 |
CN114990064B true CN114990064B (en) | 2023-07-14 |
Family
ID=83019275
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210816611.8A Active CN114990064B (en) | 2022-07-12 | 2022-07-12 | Hematopoietic stem cells, preparation method and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114990064B (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112048470A (en) * | 2020-09-17 | 2020-12-08 | 深圳丹伦基因科技有限公司 | Method for preparing clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells |
-
2022
- 2022-07-12 CN CN202210816611.8A patent/CN114990064B/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112048470A (en) * | 2020-09-17 | 2020-12-08 | 深圳丹伦基因科技有限公司 | Method for preparing clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells |
Non-Patent Citations (1)
Title |
---|
造血干细胞的发生及生物学特性;程范军;《国外医学输血及血液学分册》;第24卷(第5期);第377-380页 * |
Also Published As
Publication number | Publication date |
---|---|
CN114990064A (en) | 2022-09-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113046318B (en) | Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells | |
Tung et al. | Ex vivo expansion of umbilical cord blood for transplantation | |
KR20150126943A (en) | Methods and materials for hematoendothelial differentiation of human pluripotent stem cells under defined conditions | |
EP3828263B1 (en) | Method and kit for preparing clinical-grade mesenchymal stem cells derived from human induced pluripotent stem cells | |
Kusuma et al. | Micropattern size‐dependent endothelial differentiation from a human induced pluripotent stem cell line | |
WO2019144605A1 (en) | High performance method for differentiation of hpscs into mscs | |
CN111826348A (en) | In-vitro efficient preparation method and application of mesenchymal stem cells derived from human induced pluripotent stem cells | |
CN112226409A (en) | Method for differentiating embryonic stem cells into CD34+ hematopoietic progenitor cells | |
CN115011553A (en) | Preparation method and application of stem neural crest-derived bone marrow mesenchymal stem cells | |
CN106834223B (en) | Method for inducing differentiation of umbilical cord mesenchymal stem cells into chondrocytes | |
CN107674858B (en) | Separation medium and separation method of bone marrow endothelial progenitor cells | |
CN114787341B (en) | Method for preparing mesenchymal stem cells from human pluripotent stem cells and mesenchymal stem cells prepared thereby | |
AU2013225946B2 (en) | Method for guiding the derivation of endothelial cells from human pluripotent stem cells employing two-dimensional, feeder-free differentiation | |
CN114990064B (en) | Hematopoietic stem cells, preparation method and application thereof | |
Orlovskaya et al. | Hematopoietic differentiation of embryonic stem cells | |
WO2020027278A1 (en) | Method for producing myocardial cells | |
CN104745529B (en) | Leptin is divided into purposes and its application in hematopoietic stem/progenitor in inducing embryo stem cell | |
US9404084B2 (en) | Regulating stem cells | |
KR100981093B1 (en) | The development of human placenta mesenchymal stem cells to promote the proliferation of CD34+ hematopoietic stem cells and CD14+ monocyte from mononuclear cells from human bone marrow and cord blood | |
WO2019144606A1 (en) | Screening and differentiating method from hpscs to mscs | |
CN115992094B (en) | Method for inducing hematopoietic stem cells by low-density monolayer and application | |
CN114891737B (en) | Preparation method and application of multipotent stem cell-derived kidney mesenchymal stem cells | |
JP5751547B2 (en) | Method for forming spherical colonies | |
CN113462636B (en) | Improved method for differentiating epidermal stem cells into liver cells | |
US20210371824A1 (en) | Production of megakaryocytes in bioreactors |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |